Approximately 10 million Americans suffer from temporomandibular joint disorders (TMJD). Despite the prevalence of orofacial persistent pain amongst the population, few treatments exist for patients due to the lack of understanding about the mechanisms involved. Recently, reports have shown that patients with myofascial TMJD or chronic neck pain experience bilateral hypersensitivity in the trigeminal region. Unilateral complete Freund's adjuvant (CFA)-induced deep tissue inflammation leads to bilateral orofacial hyperalgesia in rats. Unilateral injection of the inflammatory cytokine, interleukin-12 (IL-12) into the trigeminal subnuclei interpolaris/caudalis (VIVC) transition zone also results in bilateral orofacial hyperalgesia and is attenuated with lesions to the rostral ventromedial medulla (RVM). This study proposes to investigate whether unilaterally- induced cytokine activation of VIVC neurons, the activation of RVM neurons, and the activation of contralateral VIVC neurons are necessary for the development of bilateral deep tissue orofacial hyperalgesia. Specific aim 1 will investigate whether unilateral IL-12 release in the VIVC transition zone is necessary for the development of bilateral orofacial hyperalgesia. Using a CFA-induced inflammation model of the masseter muscle, mechanical sensitivity will be tested before and after injection of IL-1 receptor antagonist into the VIVC transition zone. Specific aim 2 will investigate whether NK1-R activation in the RVM is necessary for the development of bilateral orofacial hyperalgesia. Mechanical sensitivity will be tested after intra-RVM NK1-R antagonist microinjection following inflammation. Immunohistochemistry will be used to look at changes in SP expression in various nuclei that may project to the RVM. Specific aim 3 will investigate whether NK1-R activation in the RVM leads to descending serotonergic facilitation in the contralateral VIVC. 5-HT will be depleted from neurons in the RVM using tryptophan hydroxylase-2 shRNA and 5-HT3 receptors will be blocked with specific antagonist, Y-25130, in the contralateral VIVC. Mechanical sensitivity and Fos protein expression will be measured to determine the involvement of 5-HT/5-HT3 signaling in the development of contralateral orofacial hyperalgesia. Through better understanding of the pathways and mechanisms involved in deep tissue pain facilitation to the contralateral side, novel therapies and strategies may be developed for orofacial pain disorders. ! PUBLIC HEALTH RELEVANCE: Orofacial muscle pain related to temporomandibular joint disorders affects approximately 10-12% of the population. Referred pain (secondary pain) is a common trait amongst these patients and recently referred pain at the contralateral site has been reported with increased frequency. Little is known about the mechanisms involved in secondary hyperalgesia in the trigeminal system, which greatly diminishes the efficacy and breadth of available treatments. This study will investigate possible mechanisms involved in the development of contralateral orofacial hyperalgesia after a unilateral deep tissue injury. Understanding the mechanisms involved will enable the development of various novel therapies for the treatment of orofacial pain that may increase efficacy and decrease side effects. The application may not only apply to secondary pain but also pain at the primary source of injury.